CN102165055B - Aspergillus sp. having large-scale genome duplication - Google Patents

Aspergillus sp. having large-scale genome duplication Download PDF

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CN102165055B
CN102165055B CN201080002739.6A CN201080002739A CN102165055B CN 102165055 B CN102165055 B CN 102165055B CN 201080002739 A CN201080002739 A CN 201080002739A CN 102165055 B CN102165055 B CN 102165055B
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bacterial strain
aspergillus
gene
strain
soy sauce
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CN102165055A (en
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佐藤敦史
半谷吉识
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Kitsu Kou Man K K
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
    • A23L27/50—Soya sauce
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/145—Fungal isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C12N9/2414—Alpha-amylase (3.2.1.1.)
    • C12N9/2417—Alpha-amylase (3.2.1.1.) from microbiological source
    • C12N9/242—Fungal source
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/58—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi
    • C12N9/62—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi from Aspergillus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01001—Alpha-amylase (3.2.1.1)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
    • A23L2/70—Clarifying or fining of non-alcoholic beverages; Removing unwanted matter
    • A23L2/84—Clarifying or fining of non-alcoholic beverages; Removing unwanted matter using microorganisms or biological material, e.g. enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/58—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/66—Aspergillus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/66—Aspergillus
    • C12R2001/69—Aspergillus oryzae

Abstract

Mutation was caused in a strain belonging to Aspergillus, with the goal of cultivating, without using recombinant DNA techniques, a strain of Aspergillus having improved productivity of a variety of industrially-applicable enzymes. Said mutation resulted in a strain of Aspergillus having a large-scale genome duplication of at least 900 kb, said strain having high productivity of a variety of enzymes necessary for the production of soy sauce, such as protease. This strain allows efficient production of soy sauce and various other food products.

Description

There is the aspergillus that extensive genome repeats
Technical field
The present invention relates to have the aspergillus etc. of the repetition of 900kb or above extensive genome area.
Background technology
The enzyme being produced by aspergillus has been used in multiple industry.
For example, the plurality of enzymes being produced by aspergillus is for the production of Japanese traditional food soy sauce.In order to produce soy sauce, aspergillus is grown in the soybean and wheat of raw material, and makes it produce plurality of enzymes.The enzyme decomposing protein that these are produced by aspergillus, carbohydrate, lipid etc., and in later step, promote lactic fermentation and yeast fermentation.When aspergillus produces the enzyme of a large amount of decomposition raw materials in this process, can improve utilization ratio and the compression level of raw material, significantly to boost productivity.In addition,, because the matrix of q.s is provided to lactic fermentation and yeast fermentation, fermentation is carried out aptly.Therefore soy sauce quality is significantly promoted.
Therefore,, from industrial point, the breeding with the aspergillus of high enzyme productivity is very important.For this reason, up to the present carry out energetically breeding always.Because the whole genome sequence identified (non-patent literature 1) of aspergillus oryzae (Aspergillus oryzae) RIB40, this bacterial strain is suitable for breeding.
The aspergillus breeding method that the high enzyme productivity of take is object, is roughly divided into mutation method and genetic recombination method.
Genetic recombination method comprises that with conversion method, target gene being imported to aspergillus carrys out breeding transformant.The common size of gene importing by the method is 5 to 6kb.The importing of 10kb or larger gene, because transformation efficiency significantly reduces, is therefore very difficult to successfully.In addition the region being imported into, comprises promoter region, structure gene district or the termination subarea of target gene or can in screening, be used as in some cases the gene of mark.Therefore it is difficult, in 10kb or following fragment, introducing a plurality of genes.Therefore, gene recombination method is only for example only effectively in the production of enzyme at the industrial circle that only needs High-efficient Production single enzyme, and High-efficient Production is for decomposing for example food mfg of industrial circle of a plurality of enzymes of raw material simultaneously, genetic recombination method is ineffective.The soy sauce quoted at the example as food mfg need to be produced a large amount of various enzymes in manufacturing, and comprises enzyme for improving the various raw materials of decomposition of productive rate, for improving the various enzymes of compression level, for the enzyme of enrichment savoriness etc.For by genetic recombination method while these various enzymes of High-efficient Production, should repeatedly transform.For this reason, needing to build can be by the system of the mark cycling and reutilization using in screening of recombinant.But it is extremely difficult building this system.Even if suppose to transform, repeatedly carried out and therefore the gene of the enzyme of a large amount of kinds be inserted in genome, but due to the problem relevant with expression Controlling System to locus, the gene being inserted into always can not play one's part to the full.In addition, because the unknown portions of the enzyme production mechanism of aspergillus is still a lot, so only also cannot guarantee to improve the productivity of target enzyme by inserting gene.
In addition, use gene recombination technology to carry out food mfg, be not accepted yet in the Japanese market.Therefore, genetic recombination method is applied to food mfg, has in practice some problems.Therefore, breeding is by being used for example mutating technology of ultraviolet irradiation to carry out.But the method causes the association sudden change outside targeted mutagenesis.Therefore, in this case, allow to obtain large-duty mutant with required enzyme, some problems still occur continually, such as growth velocity slowly, the productivity reduction of other enzymes etc.
In addition, aspergillus mutant is unstable in heredity in many cases.That is to say, when mutant is grown, the character of often observing mutant will become identical with the character of its parental strain (this phenomenon will be called as " reverse mutation " below), and this gives rise to trouble in industrial application.
Therefore, although need the aspergillus of the various enzymes of High-efficient Production simultaneously in industry for example needs the foodstuffs industry of plurality of enzymes simultaneously, but still there is problem discussed above.
Use mutation method the aspergillus of High-efficient Production enzyme to be carried out to the example of currently known methods of breeding as follows: to comprise and use N-methyl-N '-nitro-N-nitrosoguanidine (becoming below NTG) to process aspergillus oryzae RIB128 to there is the bacterial strain of high phytase activity to obtain, and reduce the phytic acid amount in pure mellow wine and increase the method (patent documentation 1) of the amount of biologically active substance inositol; Comprise and use NTG to process aspergillus oryzae AJ117281 to obtain the mutant strain with high protein enzymic activity, and the method (patent documentation 2) of producing the proteolysate that is rich in nitrogen; Comprise and use NTG to process aspergillus oryzae AJ117290 (FERMP-14259) to obtain the mutant strain with high glutaminase active, and produce the method (patent documentation 3) etc. of the proteolysate with high glutamic acid content.For the induction of sudden change, for the example of the medicament of induced mutation, not only comprise above-mentioned NTG, also comprise other conventional chemical substance such as azanols, ethyl-methyl sulfonic acid etc., or with irradiation such as ultraviolet ray, radioactive rays, X-rays.
Use other examples of the known breeding of mutation method to comprise: to comprise and use NTG sudden change aspergillus oryzae O-1013 (FERM P-16528) to obtain the method (patent documentation 4) of the mutant strain of the positive iron CR of High-efficient Production de-iron (Deferriferrichrysin); Comprise and use UV treatment Aspergillus sojae (Aspergillus sojae) to there is the conidial white aspergillus mutant of white to obtain, and obtain the method (patent documentation 5) of the Japanese miso with good tone; Comprise and use X-irradiation induction aspergillus oryzae for example to obtain the isovaleric aldehyde productivity with reduction, for preventing the method (patent documentation 6) as the generation of vexed (stuffy) taste of pure mellow wine deterioration odour; Comprise with Heavy Ion Beams Irradiation monascus (Monascus) and using and obtain High-efficient Production as the method (patent documentation 7) of the Aspergillus strain of the monacolin K of decreasing cholesterol material; Comprise with the iron ion bundle irradiation microorganism of giving linear energy to shift the insertion mutation of about 1.2kb and the method for deletion mutantion (patent documentation 8); Use the example (non-patent literature 2) of heavy ion beam structure Aspergillus Niger Mutant etc.
prior art document
Patent documentation
Patent documentation 1:JP-A-6-153896
Patent documentation 2:JP-A-7-274944
Patent documentation 3:JP-A-10-210967
Patent documentation 4:JP-A-2008-054580
Patent documentation 5:JP-A-7-222584
Patent documentation 6:JP-A-9-70287
Patent documentation 7:JP-A-2007-228849
Patent documentation 8:JP-A-2008-306991
Non-patent literature
Non-patent literature 1:Nature (2005) 438,1157-61
Non-patent literature 2:Food Sci.Technol.Res. (1999) 5 (2), 153-155
summary of the invention
the problem that the present invention is to be solved
The object of the invention is not use genetic recombination method to cultivate the aspergillus that wherein productivity of plurality of enzymes has been enhanced.
the means of dealing with problems
To achieve these goals; the present inventor is by for example, carrying out mutagenesis to belonging to the bacterial strain (Aspergillus sojae (Aspergillus sojae) or aspergillus oryzae (Aspergillus oryzae)) of Aspergillus (Aspergillus); and screen wherein the contiguous gene existing of target gene and the target gene highly active mutant strain that simultaneously becomes; successfully obtained and there is the aspergillus that 900kb or above extensive genome area repeat, thereby completed the present invention.
That is, the present invention relates to following [1] to [13].
[1] belong to a bacterial strain of Aspergillus (Aspergillus), described bacterial strain has the repetition of 900kb or genome area more than 900kb.
[2] bacterial strain described in [1], the bacterial strain that wherein belongs to Aspergillus is the bacterial strain that belongs to aspergillus oryzae (Aspergillus oryzae) or Aspergillus sojae (Aspergillus sojae).
[3] bacterial strain described in [1] or [2], it has the repetition of 900 to 2,400kb genome area.
[4] [1] arrives the bacterial strain described in [3] any one, and it comprises alkaline protease gene in the genome area repeating.
[5] [1] arrives the bacterial strain described in [4] any one, and it comprises alpha-amylase gene in the genome area repeating.
[6] [1] arrives the bacterial strain described in [5] any one, and it has the repetition corresponding to the genome area in the A0090003001003-A0090003001259 region of SC003 on the second karyomit(e) of aspergillus oryzae RIB40 (NRBC100959).
[7] [1] to the bacterial strain described in [6] any one, and its going down to posterity in culture experiment in wheat bran, until at least the 10 on behalf of only not demonstrating reverse mutation.
[8] [1] to the bacterial strain described in [7] any one, and it compares with parental strain that protease activity increases twice or more than twice.
[9] [1] to the bacterial strain described in [8] any one, and it compares with parental strain that alpha-amylase activity increases twice or more than twice.
[10] [1] arrives the bacterial strain described in [9] any one, and it is NITE ABP-733 (NITE BP-733) or NITE ABP-734 (NITE BP-734).
[11] [1] arrives the bacterial strain described in [10] any one, and it is by mutagenic obtained.
[12] soy sauce koji, it is by using the bacterial strain described in [1] to [11] any one to produce.
[13] soy sauce, it is by using the soy sauce koji described in [12] to produce.
invention advantage
According to the present invention, can successfully obtain the aspergillus of High-efficient Production plurality of enzymes simultaneously.Due to High-efficient Production plurality of enzymes simultaneously, described aspergillus is very useful from industrial point.In addition,, because do not use genetic recombination method, it can easily use in food mfg.Specifically, this aspergillus can be produced the enzyme (proteolytic enzyme etc.) of the various raw materials of decomposition required in manufacturing soy sauce in a large number, and this causes the remarkable increase of prepared using efficiency.
Often there is reverse mutation in known aspergillus mutant.Yet, because Aspergillus strain of the present invention has extensive genome, repeats and do not there is for example point mutation of little sudden change, so its stablizes and rare reverse mutation in heredity.Therefore, compare and can significantly improve productive rate with ordinary method.The effect that does not cause reverse mutation, because not only can improve the utilising efficiency of raw material in soy sauce manufacture, and can significantly slow down the management labour of kind of bent (song draws), goes far towards the raising of productivity, in industrial application, is therefore superior especially.
accompanying drawing summary
Fig. 1 has shown the measuring result of the protease activity of bacterial strain S1-1 of the present invention, S1-2 and S1-3 in wheat bran.
Fig. 2 has shown the measuring result of the alpha-amylase activity of bacterial strain S1-1 of the present invention, S1-2 and S1-3 in wheat bran.
Fig. 3 has shown the measuring result of the protease activity of bacterial strain R40-1 of the present invention in wheat bran.
Fig. 4 has shown the measuring result of the alpha-amylase activity of bacterial strain R40-1 of the present invention in wheat bran.
Fig. 5 has shown the go down to posterity measuring result of the protease activity in culture experiment of in wheat bran bacterial strain S1-1 of the present invention, S1-2 and S1-3.
Fig. 6 has shown the measuring result of the protease activity of bacterial strain S1-1 of the present invention, S1-2 and S1-3 in soy sauce koji.
Fig. 7 has shown the measuring result of the song digestion level of bacterial strain S1-1 of the present invention, S1-2 and S1-3 in soy sauce koji.
Fig. 8 has shown use microarray, analyzes the result of bacterial strain S1-1 of the present invention, S1-2 and S1-3 by array comparative genome hybridization method (being called below aCGH method).
Fig. 9 has shown by the result of aCGH methods analyst bacterial strain R40-1 of the present invention.
Figure 10 has shown the result of bacterial strain S1-1 of the present invention being carried out to gene copy number check analysis by quantifying PCR method.
Figure 11 has shown in wheat bran corresponding to the enzyme of the repeat region of bacterial strain S1-1 of the present invention and another kind of not measuring result of the enzymic activity of corresponding enzyme with it.
implement best mode of the present invention
The example of the aspergillus using in the present invention comprises Aspergillus sojae (Aspergillus sojae), aspergillus oryzae (Aspergillus oryzae), aspergillus niger (Aspergillus niger), Aspergillus awamori (Aspergillus awamori) etc.Wherein, the bacterial strain that belongs to Aspergillus sojae and aspergillus oryzae is preferred.
The bacterial strain that the example of these bacterial strains comprises Yi Bei preservation mechanism's preservation and easily obtains for the professional of the art, such as Aspergillus sojae 262 (FERM P-2188), Aspergillus sojae 2165 (FERM P-7280), Aspergillus sojae (ATCC42251), aspergillus oryzae (IAM2638), aspergillus oryzae RIB40 (NBRC100959) etc.
Bacterial strain of the present invention be characterised in that it have at least two 900kb that repeat copy or above, preferably from 900 to 2,400kb extensive genome area, repeat.The genome area of these repetitions is arranged on single karyomit(e), or is positioned on coloured differently body separated from one another.
In the manufacture of soy sauce, need simultaneously High-efficient Production for protein hydrolysate and produce to the contributive amino acid whose proteolytic enzyme of savoriness and for generation of at lactic fermentation and the yeast fermentation diastatic aspergillus of the glucose of thing as an alternative.Genomic data based on aspergillus oryzae RIB40, the gene of having illustrated these enzymes is positioned on same karyomit(e), by kinetochore, is not separated.By first selecting proteolytic enzyme throughput to improve mutant strain more than twice; then from mutant strain, further select amylase activity to improve twice or bacterial strain more than twice, can effectively obtain and there is the aspergillus that demonstrates the high reactivity of these enzymes simultaneously and there is the stable in properties of extensive genome area repetition.Although described as an example in this article proteolytic enzyme and diastatic combination, any combination is all possible, as long as gene is positioned on same karyomit(e) and is not separated by kinetochore.
The representative instance repeating as this extensive genome area; can should be mentioned that and stem from (corresponding to) contain alkaline protease gene and/or alpha-amylase gene and be positioned at the repetition in the SC003 region on the second karyomit(e) of aspergillus oryzae RIB40 (NBRC100959), described aspergillus oryzae RIB40 is by state-run technology and evaluation studies institute's Biological resources center (Biological Resource Center of National Institute of Technology and Evaluation (NITE)) (or the state-run institute (National Research Institute of Brewing) of brewageing) preservation." SC003 region " can by retrieval DOGAN (group of the analyzing gene database on NITE) ( http:// www.bio.nite.go.jp/dogan/Top) be appointed as ( http:// www.bio.nite.go.jp/dogan/GeneMap? GENOME_ID=ao_G2), described DOGAN is the genome analysis database of state-run technology and evaluation studies institute (National Institute of Technology and Evaluation (NITE)).In addition, SC003 region is presented at DNA Res.2008 Aug; 15 (4); In Fig. 2 of 172-83.
Alternatively, the representative instance repeating as extensive genome area, can mention as the regional on the second karyomit(e) as shown in embodiment hereinafter, comprise alkaline protease gene and alpha-amylase gene and stem from the region of Aspergillus sojae, for example the region of about 900kb (corresponding to the region of A0090003000925-A0090003001259 in aspergillus oryzae RIB40), approximately 1, the region of 500kb (corresponding to the region of A0090003001003-A0090003001556 in aspergillus oryzae RIB40), approximately 2, the region of 400kb (corresponding to the region of A0090003000654-A0090003001556 in aspergillus oryzae RIB40) and stem from 2 of aspergillus oryzae RIB40, the region of 100kb (A0090003000759-A0090003001558) (Fig. 9).
In these regions, as the repeat region using in this application, corresponding to the region of the A0090003001003-A0090003001259 of SC003 on the second karyomit(e) in aspergillus oryzae RIB40 (NRBC100959), be particularly preferred.Corresponding zone is not limited to derive from the region of aspergillus oryzae RIB40, but comprises the corresponding zone that stems from the bacterial strain that can use in the present invention.
Although whether bacterial strain of the present invention the restriction of reverse mutation occurs in not gone down to posterity and cultivating, the bacterial strain that reverse mutation does not occur is preferred.Term used herein " reverse mutation " refers to the decline that extensive genome repeats.Whether causing that reverse mutation is as described below determines.Bacterial strain is being cultivated in wheat bran after 4 days, the activity of the enzyme gene comprising in the repetition of bacterial strain (being here protease activity, amylase activity etc.) or expression amount and parental strain are being compared.Compare with parental strain and demonstrate the above bacterial strain increasing of twice or twice and be defined as not occurring the bacterial strain of reverse mutation, and the bacterial strain that demonstrates the enzymic activity that is equal to or less than parental strain is defined as occurring reverse mutation.In addition, whether bacterial strain occurs that reverse mutation is as described below to be determined.Going down to posterity in culture experiment of being undertaken by the ordinary method of use wheat bran, to at least the 10, on behalf of the bacterial strain that does not only demonstrate reverse mutation, be called as " not reverse mutation bacterial strain ", and the bacterial strain that reverse mutation occurred before the 10th generation is called as " reverse mutation bacterial strain ".
Bacterial strain of the present invention can build by known any mutafacient system, and for example NTG processes or use heavy ion beam, ultraviolet ray or X-x ray irradiation x.
For example,, in the situation that using ultraviolet irradiation, by approximately 10 of above-mentioned bacterial strains 6individual conidium is coated on casein substratum [0.4% milk casein, 0.05% casamino acids, 0.36% potassium primary phosphate, 1.43%; Sodium phosphate dibasic, 0.5% magnesium sulfate, 0.002% ferrous sulfate, 2% agar, pH6.5] upper, and flat board is used in clean bench to ultraviolet irradiation approximately 5 to approximately 10 minutes.
As the example of the method for Screening, Mutation bacterial strain, can should be mentioned that screening has the method for the bacterial strain of high protein enzyme productivity.Method comprises with irradiated Aspergillus strain inoculation casein substratum, bacterial strain is cultivated to appropriate time at the temperature that is suitable for aspergillus growth.After cultivation completes, be chosen in the bacterial strain that its periphery of bacterial colonies demonstrates large clear area, and check the character of brewageing of bacterial strain, to screen the mutant strain that demonstrates high protein enzyme productivity.
After ultraviolet irradiation, can be by for example method bacterium below.By cultivating on flat board at 30 ℃ 3 to 5 days, conidium is fixed.Then, by single bacterium colony separation selectivity collection purifying, demonstrate the bacterial strain of large clear area.Collect whole conidiums of other bacterial strains, with sterilized water, suitably dilute, be then coated on casein plate.At 30 ℃, cultivate after 3 to 5 days, by single bacterium colony separation selectivity collection purifying, demonstrate the bacterial strain of large clear area.
As the example that belongs to the bacterial strain of the present invention of Aspergillus, the R40-1 bacterial strain of describing in the S1-1 bacterial strain of describing in embodiment 1 and embodiment 2 is preserved in independent administrative corporation's product evaluation technical foundation mechanism's patent microbial preservation center (Patent Microorganism Depositary on April 6th, 2009, National Institute of Technology and Evaluation (2-5-8 Kazusakamatari, Kisarazu-shi, Chiba, 292-0818 JAPAN)), preserving number is respectively NITE P-733 and NITE P-734.In addition, for S1-1 bacterial strain and R40-1 bacterial strain, the request of submitting to the international preservation of Liao Xiang to shift to mechanism according to budapest treaty, and be accepted as respectively NITE ABP-733 and NITE ABP-734.
Hereinafter, with reference to the following examples, describe the present invention.But technical scope of the present invention is not limited by the examples.
(wheat bran preparation method)
By ordinary method, assess the enzymic activity of aspergillus.That is, upper sprinkling has the 5g Wheat bran of 80% water to install in 150ml triangular flask, and sterilizing 50 minutes at 121 ℃.Then inoculate approximately 2 platinum loop aspergillus, and at 30 ℃, cultivate 4 days.After cultivation completes, add 100ml sterilized water.Then by rubber stopper seal, shake well for culturing bottle and make its at room temperature standing 4 hours.After filtering by No. 2 filter paper (being manufactured by Advantech company), using the extracting solution obtaining as enzyme sample.
(measuring the method for protease activity)
The enzyme sample of acquisition is suitably diluted, and according in " the Shoyu Shiken-Ho method of soy sauce (chemical examination) (Methods for Testing Soy Sauce) " (Japanese soy sauce laboratory, 1985,287 pages) in the following method described measure.
The superfine TYR of 100mg is dissolved in distilled water and is settled to 1L.In 10ml volumetric flask, import respectively 1,2,3,4 and 5ml solution, and add distilled water to be settled to 10ml, to obtain the tyrosine standardized solution of 10,20,30,40 and 50 μ g/ml.In test tube, respectively import 2ml tyrosine standardized solution, and add the sodium carbonate solution of 5ml 0.55M and 1ml to dilute the Folin reagent (Folin reagent) of three times.In Water Tank with Temp.-controlled, at 30 ℃, maintain after 30 minutes, use spectrophotometer at 660nm place, to measure absorbancy.Pond, is used distilled water and carries out identical operation in contrast.Therefore, the tyrosine content (μ g) of take is X-coordinate, and 660nm place absorbancy is that ordinate zou is drawn out typical curve.Above-mentioned enzyme sample is suitably diluted, and get its 1ml and mix with 1ml 1.5% milk casein solution.At 30 ℃, react after 10 minutes, by adding the trichoroacetic acid(TCA) solution termination reaction of 3ml 0.4M.Then reaction mixture is placed 30 minutes in Water Tank with Temp.-controlled at 30 ℃, the precipitation forming is thus filtered by No. 2 filter paper (being manufactured by Advantech company).2ml filtrate is imported to test tube, and measure the absorbancy at 660nm place.As blank, before adding enzyme, add the trichoroacetic acid(TCA) solution of 3ml 0.4M and carry out same step.The difference that deducts blank absorbancy and obtain from the absorbancy of sample, is called as Δ E.Then from typical curve and Δ E, determine the amount of tyrosine.The enzyme amount that can discharge the non-proteinaceous matter that is equivalent to 1 μ g tyrosine in 1 minute is called as 1U (unit), and calculates the value of every gram of wheat bran.
(measuring the method for alpha-amylase activity)
The enzyme sample of acquisition is suitably diluted.Use α-amylase to measure test kit (Kikkoman brewages assay kit, numbering 60213), the scheme of enclosing according to test kit is measured.Alpha-amylase activity is called 1U (unit) and represents by every gram of wheat bran being discharged in 1 minute to the titre of the chloro-4-nitrophenols of 1 μ mol 2-.
(measuring the method for CMCase activity)
Use carboxymethyl cellulose (CMC) as substrate, by dinitrosalicylic acid (DNS) method, detect.1.0g substrate is dissolved in 60ml distilled water, and uses 0.4M acetic acid that pH is adjusted to 4.8.Next, add 25ml 0.4M acetate buffer (pH4.8), and with distilled water, cumulative volume is adjusted to 100ml, to obtain 1% substrate solution.To this 1% substrate solution, add equivalent amounts of enzyme sample, and carry out the reaction of 1 hour at 40 ℃.By be heated to 100 ℃ 10 minutes with termination reaction after, get 0.75ml liquid reaction mixture to test tube.To it, add 0.75ml DNS reaction reagent and by mixture shake well.Next, test tube sealed with glass stopper and boil 7 minutes.After cooling, add 3ml distilled water, and measure the absorbancy at 535nm place.The enzyme amount that can discharge the reducing sugar that is equivalent to 1mg glucose amount in 1 minute, is called as 1U (unit).
embodiment 1
By belonging to the conidium of the S1 bacterial strain (returning Kikkoman Corporation to own) of Aspergillus sojae, by carrying out mutagenesis with ultraviolet irradiation or by one of known mutagenic treatment method outside ultraviolet irradiation in the above described manner.Then conidium is coated on casein substratum, and screens according to clear area size.To demonstrate than the clear area of parental strain large 1.5 times or above bacterial strain, in wheat bran, cultivate 4 days in 30 ℃, and called after S1-1, S1-2 and S1-3 respectively.S1-1 is the bacterial strain obtaining by mutagenesis through ultraviolet irradiation, and S1-2 and S1-3 are the bacterial strains obtaining by the known mutagenic treatment method outside ultraviolet irradiation.By above-mentioned measuring method, each bacterial strain is carried out to protease activity measurement.As the result of measuring, S1-1, S1-2 and S1-3 demonstrate separately than the high twice of parental strain or protease activity more than twice and alpha-amylase activity.
Fig. 1 has shown the protease activity measuring result of each mutant strain in wheat bran.
Fig. 2 has shown the alpha-amylase activity measuring result of each mutant strain in wheat bran.
embodiment 2
The conidium of aspergillus oryzae RIB40 (NBRC100959) is carried out to mutagenesis with ultraviolet irradiation in the same manner as described above.In the same manner as in Example 1, then these conidiums are coated on casein substratum, and screen according to clear area size.By demonstrating than the clear area of parental strain large 1.5 times or above bacterial strain, in wheat bran, cultivate 4 days in 30 ℃, and called after R40-1 bacterial strain.Next, by above-mentioned measuring method, measure protease activity.As measuring result, R40-1 bacterial strain demonstrates than more than the high twice of parental strain protease activity and alpha-amylase activity.
Fig. 3 has shown the protease activity measuring result of the mutant strain obtaining in wheat bran.
Fig. 2 has shown the alpha-amylase activity measuring result of the mutant strain obtaining in wheat bran.
embodiment 3
In addition, mutant strain S1-1, the S1-2 of acquisition and S1-3 have been carried out going down to posterity culture experiment in wheat bran, and measured protease activity (Fig. 5) according to above-mentioned measuring method.As Fig. 5 is clear, show, the mutant strain of all acquisitions is until the 10th on behalf of only stably demonstrating than the protease activity of parental strain high (twice or more than twice), show that reverse mutation does not occur bacterial strain.
embodiment 4
(manufacture of soy sauce koji)
To boiling, cross every kind of above-mentioned bacterial strains that the soybean of also degreasing and the mixture of the wheat groat that bakes and pulverize add 0.1% (w/w) to draw as song, and the mixture obtaining is carried out to the koji of 3 days.Then according to measuring method above, measure the protease activity (Fig. 6) of the soy sauce koji obtaining.As Fig. 6 is clear, show, all S1-1, S1-2 and S1-3 demonstrate than the high twice of parental strain or above protease activity.
Embodiment 5
According to the following method of describing, measure the song digestion level of above-mentioned soy sauce koji in " Shoyu Shiken-Ho (method of chemical examination soy sauce) (Methods for Testing Soy Sauce) " (Japanese soy sauce laboratory, 1985,104 pages).
The song digestion level of measurement soy sauce koji as described below.In 500ml triangular flask, pack every kind of soy sauce koji of 100g into, and add the sodium chloride aqueous solution of 200ml 18.5% to it.Shaking flask is sealed with cork stopper, then at 37 ℃, carry out the self-digestion of 1 week.During this period, the mixture in shaking flask is stirred once every day.The sauce wine with dregs (mash) of so digestion is evenly mixed in mixing tank, and measure total nitrogen content and the sodium chloride content of sauce wine with dregs.In addition, the sauce wine with dregs stirring evenly is filtered, and also measure total nitrogen content and the sodium chloride content of filtrate.From take off data, determine according to the following formula digestion level (Fig. 7).As Fig. 7 is clear, show, S1-1, S1-2 and S1-3 demonstrate than S1, are the digestion level that parental strain is higher.The raising of bent digestion level directly causes soy sauce to manufacture the raising of the utilising efficiency of Raw.
Bent digestion level (%)=[(the NaCl content % in sauce wine with dregs)/(the total N content % in sauce wine with dregs)] * [(the N content % in filtrate)/(the NaCl content % in filtrate)] * 100
embodiment 6
(using microarray, the analysis of being undertaken by array comparative genome hybridization method (aCGH method))
Use microarray (Agilent Technologies), by aCGH method analyzing in detail the copy number changes of S1-1 bacterial strain.Found that; the gene existing in the approximately 900kb region that comprises alkaline protease gene and alpha-amylase gene (corresponding to A0090003000925-A0090003001259 in aspergillus oryzae RIB40) compares increase twice with parental strain or more than twice, this shows that S1-1 bacterial strain has extensive genome and repeats.Also by aCGH, other proteinase high-yield bacterial strains have been analyzed.Found that; S1-2 has approximately 1; the extensive genome in the region of 500kb (corresponding to the region of A0090003001003-A0090003001556 in aspergillus oryzae RIB40) repeats; S1-3 has approximately 2; the extensive genome in the region of 400kb (corresponding to the region of A0090003000654-A0090003001556 in aspergillus oryzae RIB40) repeats, wherein each district inclusion alkaline protease gene and alpha-amylase gene (Fig. 8).
Equally, by aCGH methods analyst the R40-1 bacterial strain obtaining in embodiment 2.Found that, this bacterial strain and S1-1 bacterial strain are similar has approximately 2, and the extensive genome in the region that comprises alkaline protease gene and alpha-amylase gene (A0090003000759-A0090003001558) of 100kb repeats (Fig. 9).
embodiment 7
(the alkaline protease gene copy number check analysis of being undertaken by quantifying PCR method)
By quantifying PCR method quantitative the gene copy number of each gene in the repeat region of S1-1 bacterial strain, and with parental strain S1 in compare (M * 3000P, Stratagene) (Figure 10).PCR carries out 40 circulations, and each circulation forms by 95 ℃ 10 seconds, 60 ℃ for 20 seconds and 72 ℃ for 15 seconds.Gene in contrast, has been used flbA gene in the brlA gene in the first chromosome, trisome and the pceB gene in tetrasome.As Figure 10 knows demonstration, S1-1 bacterial strain is compared with parental strain S1, be present in by aCGH method and turn out to be alkaline protease gene (alp), amylase gene (amy) and the pceA gene in the approximately 900kb region of repetition, its amount of every kind, up to twice, has been verified the analytical results of aCGH method thus.The base sequence of the primer using is described below (table 1), i.e. brlA-F (SEQ ID NO:1), brlA-R (SEQ ID NO:2), alp-F (SEQ ID NO:3), alp-R (SEQ ID NO:4), amy-F (SEQ ID NO:5), amy-R (SEQ ID NO:6), pceA-F (SEQ ID NO:7), pceA-R (SEQ ID NO:8), flbA-F (SEQ ID NO:9), flbA-R (SEQ ID NO:10), pceB-F (SEQ ID NO:11) and pceB-R (SEQ ID NO:12).Primer sequence not only can be used for Aspergillus sojae, and can be used for using the copy of the range gene in repeat region in bacterial strain in this application.
Table 1: for the primer sequence of quantitative PCR
Figure BPA00001332482800151
embodiment 8
Use S1-1 bacterial strain to prepare wheat bran, and by aforesaid method, measure the activity of every kind of enzyme.Result, the activity of the enzyme of the genes encoding comprising in genome repeats (proteolytic enzyme and α-amylase) is compared remarkable increase with parental strain, increase twice or more than twice, and carboxymethylcelluloenzyme enzyme (CMCase) activity not being included in aforementioned region does not demonstrate considerable change (Figure 11).Result shows, the increase of proteolytic enzyme and alpha-amylase activity depends on repetition.
Although the present invention be have been described in detail by reference to specific embodiment, the professional of the art should be appreciated that, can make various modifications and change and not deviate from the spirit and scope of the present invention.
The application is the Japanese patent application (Japanese patent application 2009-100645) of submitting to based on April 17th, 2009, and its disclosure is incorporated by reference at this.
industrial applicibility
By by the inoculation that belongs to Aspergillus of the present invention in raw materials of soy sauce and cultivate, can obtain solid koji and liquid koji that the throughput of plurality of enzymes improves.Use described song to produce soy sauce with mash.By by inoculation of the present invention in rice raw material and cultivate; Can produce fermented glutinour rice, fermented glutinour rice series products and pure mellow wine.In addition,, by the liquid culture of bacterial strain of the present invention, can produce the sauce that comprises digested gluten.Therefore, the present invention relates to such production method and the various food produced thus soy sauce for example.
the open text of sequence table
SEQ ID NO:1 represents the primer sequence for brlA gene.
SEQ ID NO:2 represents the primer sequence for brlA gene.
SEQ ID NO:3 represents the primer sequence for alp gene.
SEQ ID NO:4 represents the primer sequence for alp gene.
SEQ ID NO:5 represents the primer sequence for amy gene.
SEQ ID NO:6 represents the primer sequence for amy gene.
SEQ ID NO:7 represents the primer sequence for pceA gene.
SEQ ID NO:8 represents the primer sequence for pceA gene.
SEQ ID NO:9 represents the primer sequence for flbA gene.
SEQ ID NO:10 represents the primer sequence for flbA gene.
SEQ ID NO:11 represents the primer sequence for pceB gene.
SEQ ID NO:12 represents the primer sequence for pceB gene.
Figure IPA00001332482300011
Figure IPA00001332482300031

Claims (8)

  1. Deposit number be NITE BP-733 Aspergillus sojae ( aspergillus sojae) S1-1 bacterial strain or deposit number be NITE BP-734 aspergillus oryzae ( aspergillus oryzae) R40-1 bacterial strain, it has the repetition corresponding to the genome area in the A0090003001003-A0090003001259 region of SC003 on the second karyomit(e) of aspergillus oryzae RIB40.
  2. 2. bacterial strain claimed in claim 1, it has the repetition of 900 to 2,400kb genome area.
  3. 3. bacterial strain claimed in claim 1, it comprises alkaline protease gene in the genome area repeating.
  4. 4. bacterial strain claimed in claim 2, it comprises alkaline protease gene in the genome area repeating.
  5. 5. the bacterial strain described in claim 1 to 4 any one, it comprises alpha-amylase gene in the genome area repeating.
  6. 6. the bacterial strain described in claim 1 to 4 any one, its routine at wheat bran goes down to posterity in culture experiment, until at least the 10 generation did not demonstrate reverse mutation.
  7. 7. bacterial strain claimed in claim 5, its routine at wheat bran goes down to posterity in culture experiment, until at least the 10 generation did not demonstrate reverse mutation.
  8. 8. a soy sauce koji, it requires the bacterial strain described in 1 to 7 any one to produce by right to use.
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